Figure 5.

ALK1 binds to TGF-β1 in ECs, activates Smad1 and Smad5, and can inhibit the TGF-β1-responsive 3TP-Lux induction. (A) TGF-β1 binds to ALK1 as well as ALK5 (TβR-I) in HUVEC. HUVEC were affinity crosslinked with ¹²⁵I-TGF-β1 and immunoprecipitated by using antibodies specific to either ALK1 or ALK5. Blocking of ALK1 antiserum was done in the presence of the peptide used for immunization of the rabbit (15). The ALK1 band migrated slightly slower than ALK5, in agreement with the difference in the sizes of ALK1 and ALK5. (B) ALK1 phosphorylates Smad1 and Smad5 but not Smad2. COS cells were transfected with Flag-tagged (F) Smad1, Smad5, or Smad2, and constitutively active (c.a.) forms of ALK1 (A1) or ALK5 (A5). (Top). Phosphorylation of Smads was detected by immunoprecipitation of each Smad with a Flag antibody followed by immunoblotting by using an antiphosphoserine antiserum. (Center and Bottom). Expression of Smads and ALKs was determined by anti-Flag and antihemagglutinin antibodies, respectively. (C) ALK1 inhibits TGF-β1-dependent 3TP-Lux induction mediated by ALK5 in HepG2 cells. A TGF-β responsive element linked to the luciferase gene (3TP-Lux) was transfected into HepG2 cells together with increasing amounts of ALK1 or ALK2 in the presence of ALK5. After transfection, cells were treated with (solid bars) or without (open bars) TGF-β1 (5 ng/ml) for 24 hr. Luciferase activities were then measured and plotted. In all assays, luciferase activities are plotted in arbitrary units. (D) The balance model for TGF-β1 signaling in regulation of angiogenesis. Explanation is in the text.